PROJECT SUMMARY/ABSTRACT?Transgenic Gene Knockout Shared Resource Genetically modified mice are invaluable for SJCCC researchers to evaluate gene function in vivo and determine how specific genes regulate cell growth and differentiation in normal and tumor tissue. The Transgenic Gene Knockout Shared Resource (TGKSR) centralizes services for the generation of genetically modified mice and provides education, expertise, and cost effectiveness that would be extremely difficult to maintain in individual laboratories. The TGKSR Director is Hartmut Berns, PhD, an expert in transgenic technology with 23 years' experience creating genetically engineered mice, 16 years thereof in the management of transgenic cores at Comprehensive Cancer Centers. He is supported by 5 full-time technologists who employ state-of-the-art gene- modification technologies to produce transgenic mice by pronucleus injection of DNA into zygotes, ESC-based gene-targeted mice by injection of genetically engineered ESCs into blastocyst-staged embryos, and gene- edited mice. Engineered mice are also generated by injection of the endonucleases TALEN or CRISPR/Cas9 into the pronucleus or cytoplasm of zygotes to produce gene KOs, knock-ins (KIs), conditional genes, or point mutations. There has been marked impact of the TGKSR on the science of SJCCC members. Researchers have gained insights from germline-transmitting chimeras of 41 different ESC-based targeted genes, and multiple founders of 34 DNA constructs during the current funding cycle. The TGKSR was used by 18 investigators of whom 83% (n=15/18) are members of the Cancer Center. Of these, 93% (n=14/15) hold cancer- focused peer-reviewed grants. These SJCCC members were drawn from 4 of 5 SJCCC Programs and yielded a combined total of 57 publications from CBP (n=26), NBTP (n=22), HMP (n=13), and DBSTP (n=1). During the upcoming cycle, in recognition of the growing impact of CRISPR/Cas9 reagents on advancing research performed by the SJCCC, the TGKSR will further enhance interactions with the new Center for Advanced Genome Engineering (CAGE) Shared Resource. We will create standardized workflows for bringing reagents designed by CAGE into the TGKSR and, in turn, for validating GEM tissue samples by CAGE in order to optimize GEM production. We will carry out pilot projects to test reagent modifications and concentrations to identify the most efficient and effective CRISPR approaches by comparing the efficiencies of pronuclear injections and cytoplasmic injections in zygotes. Our goal is to optimize these new reagents and their application in zygotes (to generate KOs, small KIs, point mutations, conditional alleles) and in ESCs (to generate large KIs and more involved conditional and inducible alleles) to most efficiently provide GEM to SJCCC members. Additionally, microinjection, surgery, and animal rooms of the TGKSR are slated to move to a new vivarium to be constructed on the St. Jude campus, with ground-breaking slated for spring 2018 and opening for 2021. Thorough planning of the scope and size of microinjection capabilities and animal space has been initiated to ensure that the future needs of SJCCC members for GEM production are met.